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. 2016 Sep 20;113(38):E5655-64.
doi: 10.1073/pnas.1603020113. Epub 2016 Sep 6.

TRiC Subunits Enhance BDNF Axonal Transport and Rescue Striatal Atrophy in Huntington's Disease

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Free PMC article

TRiC Subunits Enhance BDNF Axonal Transport and Rescue Striatal Atrophy in Huntington's Disease

Xiaobei Zhao et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Corticostriatal atrophy is a cardinal manifestation of Huntington's disease (HD). However, the mechanism(s) by which mutant huntingtin (mHTT) protein contributes to the degeneration of the corticostriatal circuit is not well understood. We recreated the corticostriatal circuit in microfluidic chambers, pairing cortical and striatal neurons from the BACHD model of HD and its WT control. There were reduced synaptic connectivity and atrophy of striatal neurons in cultures in which BACHD cortical and striatal neurons were paired. However, these changes were prevented if WT cortical neurons were paired with BACHD striatal neurons; synthesis and release of brain-derived neurotrophic factor (BDNF) from WT cortical axons were responsible. Consistent with these findings, there was a marked reduction in anterograde transport of BDNF in BACHD cortical neurons. Subunits of the cytosolic chaperonin T-complex 1 (TCP-1) ring complex (TRiC or CCT for chaperonin containing TCP-1) have been shown to reduce mHTT levels. Both CCT3 and the apical domain of CCT1 (ApiCCT1) decreased the level of mHTT in BACHD cortical neurons. In cortical axons, they normalized anterograde BDNF transport, restored retrograde BDNF transport, and normalized lysosomal transport. Importantly, treating BACHD cortical neurons with ApiCCT1 prevented BACHD striatal neuronal atrophy by enhancing release of BDNF that subsequently acts through tyrosine receptor kinase B (TrkB) receptor on striatal neurons. Our findings are evidence that TRiC reagent-mediated reductions in mHTT enhanced BDNF delivery to restore the trophic status of BACHD striatal neurons.

Keywords: BACHD mouse model; BDNF transport; Huntington’s disease; TRiC chaperonin; striatal atrophy.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Striatal neuron atrophy and reduced synaptic connectivity in HD are recapitulated in corticostriatal microfluidic chambers of BACHD neurons. (A) Schematic drawing of corticostriatal coculture plated in the microfluidic chamber. (B) Tau (green) and DARPP32 (red) staining in the corticostriatal coculture. (C) Experimental time course. (D) Analysis of soma sizes of cortical and striatal neurons in WT-WT, BACHD-BACHD, and WT-BACHD cocultures. Results are shown as mean ± SD from two independent chambers with n = 10 fields and a total of 50–100 cells. ***P < 0.001 using Dunnett’s post hoc test of multiple comparisons to the WT-WT striatal soma size. (E) Synaptophysin (green) and PSD95 (red) staining in the striatal compartment. (F) Comparison of pre- and postsynaptic marker colocalization using Spearman’s rank correlation. Results are shown as mean ± SD from two independent chambers with n = 10 fields and 50–100 cells. *P < 0.05 using Dunnett’s post hoc test of multiple comparisons to the WT-WT group striatal soma size.
Fig. 2.
Fig. 2.
Effect of BDNF on the trophic status of BACHD striatal neurons. (A) Experimental time course for studies of soma size and trafficking. (B) Analysis of soma sizes of striatal neurons in WT and BACHD cultures treated with BDNF. Results are shown as mean ± SD from two independent chambers with n = 10 fields and a total of 50–100 cells. Significant differences, ***P < 0.001, were determined by Bonferroni post hoc comparisons of selected pairs. n.s., not significant. (C) Analysis of soma sizes of cortical and striatal neurons in WT-WT and WT-BACHD cocultures with cortical neurons treated with BDNF siRNA or mismatch siRNA. Results are shown as mean ± SD from two independent chambers with n = 10 fields and a total of 50–100 cells. *P < 0.05 and ***P < 0.001 were determined using Dunnett’s post hoc test of multiple comparisons to the WT-WT striatal soma size. (D) Analysis of soma sizes of striatal neurons in WT-WT and WT-BACHD cocultures treated with BDNF antibody in striatal compartment. Results are shown as mean ± SD from two independent chambers with n = 10 fields and a total of 50–100 cells. ***P < 0.001 using Dunnett’s post hoc test of multiple comparisons to the WT-WT group striatal soma size. (E) Representative kymographs of BDNF-eGFP anterograde transport in WT and BACHD cortical neurons expressing mCherry.
Fig. S1.
Fig. S1.
Cell viability assay in WT and BACHD striatal neurons. No significant difference was detected with Student’s t test. n.s., not significant.
Fig. S2.
Fig. S2.
BDNF knockdown in 293 cells. (A) Live imaging of 293 cells coexpressing BDNF-eGFP with either the BDNF siRNA or the mismatch siRNA. (B) Twenty-four hours after BDNF knockdown, 293 cells were lysed. Lysates were analyzed by Western blotting and blotted with anti-GFP.
Fig. 3.
Fig. 3.
CCT3 and ApiCCT1 decrease mHTT in BACHD cortical neurons. (A) Experimental time course studies for measuring soma size and intensity of mHTT immunofluorescence. (B) Immunostaining of BACHD cortical neurons overexpressing CCT3 (green) with antipolyglutamine MAB1574 antibody (red). (C) Immunostaining of BACHD cortical neurons treated with ApiCCT1 with antipolyglutamine MAB1574 antibody (red). (D) Analysis of polyglutamine intensity in BACHD neurons expressing CCT3 or treated with ApiCCT1. a.u., arbitrary units. (E) Analysis of cortical neuron soma size in CCT3-treated or ApiCCT1-treated cells versus untreated cells. In D and E, results are shown as mean ± SD from three independent chambers with n = 39 for control cells, n = 33 for CCT3-positive cells, and n = 59 for ApiCCT1-treated cells. ***P < 0.001 using Dunnett’s post hoc test of multiple comparisons to the control groups. (F) Immunoblotting (IB) of mHTT using MAB1574 antibody in BACHD cortical neurons treated with CCT3 lentivirus [multiplicity of infection (MOI) of 5 and 10] and exogenous ApiCCT1 (1 or 2.5 μM). Results are shown as mean ± SD from three independent experiments. *P < 0.05 and **P < 0.01 using Dunnett’s post hoc test of multiple comparisons to the nontreated control group. (G) ApiCCT1 was detected inside primary cortical neuron cytosol and nucleus following PK treatment. Lanes are labeled as follows: C, cytoplasmic fraction; N, nuclear fraction; −PK, no PK treatment; +PK, with PK treatment; PK + ctrl, 60 μg of lysed nuclear fraction treated with PK; T, total lysate. Samples were analyzed by Western blot with anti-His antibody.
Fig. S3.
Fig. S3.
Single CCT-mCherry subunits were expressed in (A) PC12 cells and (B) rat cortical neurons.
Fig. S4.
Fig. S4.
Expression of individual CCT subunits decreased mHTT level, but not wtHtt level, in PC12 cells. (A) In PC12 cells, wtHttQ25-GFP was coexpressed with each CCT subunit. The level of wtHttQ25-GFP was not altered by expression of any CCT subunit. (B) In PC12 cells, mHTTQ97-GFP was coexpressed with each CCT subunit. Compared with the mCherry control, the level of mHTTQ97-GFP was reduced by expression of several CCTs, especially CCT1, CCT3, CCT5, CCT7, and CCT8.
Fig. S5.
Fig. S5.
CCT3 and ApiCCT1 treatments did not change the mRNA levels of either mouse wtHtt or human mHTT. Levels of mRNAs in DIV7 BACHD cortical neurons were assessed using real-time RT-PCR. Relative to untreated cells, no significant changes were observed in neurons expressing CCT3 or treated with ApiCCT1. Results are shown as mean ± SD from three independent experiments. Bonferroni post hoc comparisons of selected pairs were used to test for significance of differences.
Fig. 4.
Fig. 4.
Rescue of impaired anterograde transport of BDNF in BACHD cortical neurons by expression of CCT3 or treatment with ApiCCT1. (A) Experimental time course for studies measuring soma size and trafficking. (B) Representative kymographs of BDNF-eGFP anterograde transport in BACHD cortical neurons overexpressing CCT3 or treated with ApiCCT1. (C) Comparison of the anterograde instantaneous velocity, percentage of pause events, pause duration, and average velocity of BDNF-eGFP in WT or BACHD cortical neurons overexpressing mCherry (as a control), overexpressing CCT3-mCherry, or treated with ApiCCT1. Results are shown as mean ± SEM from three independent experiments with 30–40 movies collected and 50–75 BDNF signals recorded. Significant differences, *P < 0.05, **P < 0.01, and ***P < 0.001, were determined by Bonferroni post hoc comparisons of selected pairs. (D) Analysis of soma size of BACHD striatal neurons in BACHD-BACHD coculture when cortical neurons were treated with ApiCCT1, BDNF, or both; also shown are the effects of treatment with 10 μM ANA-12. Results are shown as mean ± SD from two independent chambers with n = 10 fields and total 60–200 cells. ***P < 0.001 using Bonferroni post hoc comparisons of selected pairs. (E) Analysis of some size of striatal neurons in WT-WT, WT-BACHD, and BACHD-BACHD cocultures treated with ApiCCT1, neutralizing BDNF antibody, or both. Results are shown as mean ± SD from two independent chambers with n = 10 fields and a total of 50–120 cells. Significant differences, **P < 0.01 and ***P < 0.001, were determined by Bonferroni post hoc comparisons of selected pairs.
Fig. S6.
Fig. S6.
ANA-12 pretreatment in striatal neurons reduced levels of pTrkB. Embryonic day 18 rat striatal neurons were treated with 10 μM ANA-12 at DIV5 for 48 h. The neurons were then treated with 100 ng/mL BDNF for 0, 5, and 30 min. Lysates were generated, and Western blotting was performed with a rabbit antibody that specifically recognizes pY490 on TrkB.
Fig. 5.
Fig. 5.
Rescue of impaired retrograde transport of BDNF in BACHD cortical neurons by overexpressing CCT3 or treating with ApiCCT1. (A) Schematic drawing showing that neurons loaded in the cell body compartment extended axons into the axon compartment. QD-BDNF was added to the axon compartment. Diffusion of the QD-BDNF to the cell body compartment was minimized by reducing the height of the medium level in the axon compartment. (B) Experimental time course for measuring QD-BDNF retrograde trafficking in DIV7 cortical neurons. (C) Representative kymographs of QD-BDNF retrograde transport in DIV7 cortical neurons from WT, BACHD, BACHD treated with CCT3, and BACHD treated with ApiCCT1. (D) Comparison of the retrograde instantaneous velocity, percentage of pause events, pause duration, and average velocity of QD-BDNF in DIV7 WT or BACHD cortical neurons that overexpressed CCT3, were treated with ApiCCT1, or were not treated. Results are shown as mean ± SEM from three independent experiments with 30–40 movies collected and 40–75 BDNF signals recorded. Significant differences, *P < 0.05 and ***P < 0.001, were determined by Bonferroni post hoc comparisons of selected pairs. (E) Experimental time course for measuring QD-BDNF retrograde trafficking in DIV4 cortical neurons. (F) Comparison of the retrograde instantaneous velocity, pause duration, and average velocity of QD-BDNF in DIV4 WT or BACHD cortical neurons. Results are shown as mean ± SEM from two independent experiments with 25–30 movies collected and 35–50 BDNF signals recorded. *P < 0.05 using Student’s t test.
Fig. S7.
Fig. S7.
Impaired lysosome transport in BACHD cortical neurons was rescued by expression of CCT3 or CCT5. (A) Live imaging of cortical neurons grown on coverslips that were transfected with CCT3 or CCT5 in a pLenti vector, which carries a GFP signal (green). At 24 h after transfection, LysoTracker (red) was added for 30 min; it was removed by washing before live imaging. (B) Representative kymographs of lysosome movement, showing both retrograde or anterograde transits, from WT, BACHD, and BACHD neurons that express CCT3 or CCT5. (C) Analysis of the instantaneous velocity of lysosomal transport in cortical neurons from WT-, BACHD-, and BACHD-treated CCT3 or CCT5 neurons. Results are shown as mean ± SEM from three independent experiments with 30–40 movies collected and 100–150 BDNF signals recorded. ***P < 0.001 using Dunnett’s post hoc test of multiple comparisons to the WT-WT group.
Fig. S8.
Fig. S8.
Expression of a single CCT subunit decreases the insoluble mHTT level in 14A2.6 cells. In 14A2.6 cells, mHTTQ103-GFP production was PA-induced after individual CCT subunits were expressed for 48 h. The level of insoluble, but not soluble, mHTTQ103-GFP was decreased.
Fig. 6.
Fig. 6.
Expression of CCT3 decreases mHTTQ103 and the number of inclusion bodies in 14A2.6 cells. (A) Live imaging of 14A2.6 cells transfected with CCT3 (red) or mCherry (red) for 48 h and then induced to produce mHTTQ103-GFP (green) using PA. (Upper) White arrows show that mHTTQ103 inclusion bodies were found in the cells that express mCherry. (Lower) Yellow arrows show that many fewer inclusion bodies were present in cells that expressed CCT3. (B) Filter trap assay of SDS-resistant mHTTQ103 aggregates using EM48 antibody in PA-induced 14A2.6 cells that were not transfected, were transfected with mCherry, or were transfected with CCT3. Results are shown as mean ± SD from five independent experiments. *P < 0.05 using Dunnett’s post hoc test of multiple comparisons to the control group. (C) Fluorescence-activated cell sorting (FACS) of 14A2.6 cells that express CCT3-mCherry or mCherry alone. Cells were sorted by fluorescence for Cy5 (due to expression of either mCherry or CCT3-mCherry) and FITC (mHTTQ103-GFP). FSC-A, forward scatter-area; P2, Cy5-positive cells; P3, Cy5-negative cells; PerCP, peridinin-chlorophyll protein. Percentage of FITC mean fluorescence intensity in P2 to P3, normalized by transfection efficiency and expressed relative to the mCherry control. Results are shown as mean ± SD from three independent experiments. *P < 0.05 using Student’s t test.
Fig. S9.
Fig. S9.
Expression of CCT5 decreased the number of inclusion bodies in 14A2.6 cells. Live imaging of 14A2.6 cells transfected with CCT5 for 48 h and then PA-induced to produce mHTTQ103-GFP is shown. Yellow arrows show that fewer inclusion bodies were present in cells that expressed CCT5.
Fig. S10.
Fig. S10.
Expression of CCT5 decreases mHTTQ103 level in 14A2.6 cells. (A) FACS of 14A2.6 cells that expressed CCT5-mCherry. Cells were sorted by fluorescence for Cy5 (due to expression of either mCherry or CCT5-mCherry) and FITC (mHTTQ103-GFP). FSC-A, forward scatter-area; P2, Cy5-positive cells; P3, Cy5-negative cells; PerCP, peridinin-chlorophyll proteins. (B) Percentage of FITC mean fluorescence intensity (MFI) in P2 to P3, normalized by transfection efficiency and expressed relative to the mCherry control. Results are shown as mean ± SD from three independent experiments. **P < 0.01 using Student’s t test.
Fig. 7.
Fig. 7.
Coimmunoprecipitation of Htt with CCT subunit(s) and the effect of proteasome inhibitor MG132 on mHTT degradation. (A) Immunoprecipitation (IP) of GFP in GFP-, wtHttQ25-GFP–, or mHTTQ97-GFP–transfected 293 cells showed that wtHttQ25 and mHTTQ97 interacted with at least several CCT subunits, Hsp70, and Hsp40. (B) The 293 cells were transfected with CCT3-mCherry or mCherry for 48 h; cycloheximide (CHX) was added for 0, 1, or 2 h. Cell lysates were collected and resolved on SDS/PAGE. (C) Immunostaining with antipolyglutamine antibody MAB1574 (red) of MG132-treated BACHD cortical neurons overexpressing CCT3 (green). Results are mean ± SD from two independent experiments with n = 38 for CCT3-positive cells, and n = 26 for CCT3-negative cells; comparison is with data (first two bars) shown in Fig. 3D. ***P < 0.001 using Dunnett’s post hoc test of multiple comparisons to the control group without MG132.

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